Thiocornine and thiamamidine phosphoric cysteine

ABSTRACT

Thiocornine, an amino acid, and thiamamidine phosphoric cysteine are obtained from vegetable sources, e.g. Allium plants, or animal sources. The compounds are therapeutically useful.

United States Patent [72] inventors Kiyoshi Kominato;

Yutaka Kominato, both 0! No. 15, Hagigakakiuchico, Shimokamo, Sakyoku,Kyoto-shi, Japan [21 Appl. No. 773,294

[22] Filed Nov. 4, 1968 [45] Patented Dec. 7, 1971 [32] Priority Nov. 8,1967 [33] Japan [54] THIOCORNINE AND THIAMAMIDINE PHOSPHORIC CYSTEINE 4Claims, No Drawings [52] U.S.Cl. 260/534, 260/302, 260/527 [5 1] Int. ClC07d 91/32 [50] Field of Search 260/2565, 534 S [56] References CitedUNITED STATES PATENTS 3,355,488 11/1967 Restivo et al. 260/534 PrimaryExaminer-Alex Mazel Assistant Examiner-R. J. GallagherAlrorneyWenderoth, Lind & Ponack ABSTRACT: Thiocornine, an amino acid,and thiamamidine phosphoric cysteine are obtained from vegetablesources, e.g. Allium plants, or animal sources. The compounds aretherapeutically useful.

THIOCORNINE AND THIAMAMIDINE PHOSPHORXC (The closed ring formula of'I.P.C.)

CYSTEINE CH: CHzCNzOH NH JH/ The present invention provides a processfor separately preparing (a) a new amino acid, thiocomine, which is usedas 5 z- SE a material for synthesizing an availablercomponent of garlicor =0 CHZCO OH\C=O for biosynthesis and (b) thiarnarnidine phosphoriccysteine L I which is used as a material for synthesizing methionine orfor biosynthesis, and the process has thecharacteristic features C 2that the starting materials include animals and plants in (The open ringformula of '1.P.C.) general, especially genus Allium plants, beer yeast,and the pressed cake of general fermentation products of plant andinmlocPfmne "l y occurs In ture In the protein part ternal organs ofanimals, which are treated (grease is removed f F fi i' as a compofem Fof beforehand in case of animal organs) with methanol (or tides, andthrocornme IS isolated by hydrolysis. It is very unstaethanol) toextract effective components and the extract is me P the solated thusdecompo? homo concentrated by evaporation, the the ingredient iscysteine and urea as s expressed by the following scheme: hydrolyzed bybeing heated with added sulfuric acid in per se conventional manner, andsubsequently it is brought into an 7 i p aqueous solution after the acidhas been removed, and then il3 the thiamamidine phosphoric cysteine isadsorbed completely NH on added active charcoal while the thiocomineremains in the 2 solution, the former being desorbed from the charcoalwith methanol or ethanol and the eluant being evaporated into NHa+0=C+HOOCCHCH:CHSH syrup form to obtain a concentrated liquid, and thefiltrate H2 1 containing the latter component being made alkaline with ahomoc stem suitable base, such as ammonia, barium hydroxide or caustic yalkali, and kept standing in a cool place for a while with a littleethanol added until a precipitate is formed to which enough The compoundthiocomine, however, is stable in the form hydrochloric acid iS added 0make the SOlUtlOIl acid [0 form of peptide or hydrochloride, [1 hasbecome evident that homocrystals of thiocomine in a short time.cysteine, which is seldom found in nature, is an important sub- Themoiety of the new amino acid, thiocomine, which is stance when theessential amino acid methionine is formed in represented by structuralformula below, is found extensively the human bodyfrom substances such:as choline or betaine in in nature as a component of the thiamamidinederivatives, the presence of methyl transfer enzyme. Consequently, thewhich presumably belong to an enzymatic system or to a new thiocorninemoiety is considered to be the only origin of vitamin, along withthiamamidine phosphoric cysteine (abhomocysteine in nature. breviatedhereinafterasT.P.C.). Further T.P.C., to which discovery the presentinventors NH devoted themselves for as longas 45 years, is a fundamentalH CH H H H Q functional group in the composition of thiamamidine deriva-2-5-1: tives and is expressed bythe molecular formula shown above. NH:The distribution of the compound moietyin nature in the form thiocommeof thiamamidine derivatives covers almost all foods and plays (I333 animportant role in the taste and nutrition of the foods. For H=CHOH thesake of illustration, the chemical structural formulas are l shown belowof scordinines as an effective component of garlic. Meanwhile there arevarious kinds of scordinines, such as H0 1"= CH-S scordinine A scordinine A etc., depending on the number NH and the order of the aminoacids in the composition of pop nooc-t m-cmsn CHzOH cu s-( --(|3H-N- (EHH0511 I HO-CH ll A 0 0 A ONE: OH H Gill-J L HO H 20 in H IHz O 0 081/2(IIJH2 emanate HCNH2 CHZSH NH HOI|=O 30NH- H OH2CH2SNHP3NH2 O CH: o-1 =0C0NH--CH I CHzCOOH (SH-S moiety of thiamamidine phosphoric cysteine (anew compound T.P.C.)

scordlnlne A scordinine B 1. Animals tested: Male and female mice ofdd-series were employed. They were bred in a breeding chamber at 23 C.

Further, the present invention substance thiocomine is also with solidfood and city water, but they were kept without useful for fat removalof fatty livers and for strengthening food and water for 3 hours beforethe administration of livers, and thiamamidine phosphoric cysteine actsas cofactor 5 thiocomine. After the administration symptoms in general(just like vitamin), and each of the substances can be adwere observedand as for dead individuals visual observation ministered to patients byhypodermic or venous injection in an of organs was carried out atdissection. The LD value was amount of 50 mg. X 2 per day or orally inan amount of I calculated, according to the Behrens-Karber method, fromthe mg. X 2 per day. number of mice that died within days.

The animal test of thiocomine rehindrance effect of fatty 1O 2.Administration and dose: Thiocornine was given to male liver formationwas made as described infra. and female mice by intraabdominal injection(i.p.). Solutions prepared by dissolving 100, 80, 60, 40 and 20 mg. perin- METHOD OF THE TEST dividual of thiocomine each in 1 ml. ofphysiological salt solu- Thirty male albino rats, each about 100 g. inweight, were 5 tion ,injected' q f f to whom I of separated into threegroups. Each group contained rats. phylslologlcalsah 501M101! W85 painly Injected. The first group was controlled, the second was the fattyliver members were used to each group ofdlfierem doses and group, andthe third was the thiocomine dosage group. (After three members forcontrol the test more healthy fine rats were chosen from each group,RESULTS and hver fat was measured.)

Compound feed and water were given to the control group; Approximately30 minutes after the intra-abdominal injecfeed containing orotic acid inthe ratio of 2 percent by weight ti n first general symptoms appeared,such as inactivated moand water were given to fatty liver group and thethiocomine tion, erected hairs, increased respiration frequency, etc.and group. More than mg. of thiocomine (dissolved in 0.5 ml. further themore the dose, the more remarkable the sympof water) was injectedintraperitoneally every day. Twenty 25 toms. Death was first observed 8hours after the administradays after the test of the thiocomine grou therat w r tion and continued for 5 days, meaning the inclination ofkilled, blood recovered, liver picked out, and fat weighed and delayedeffectiveness of thiocomine. The rest of mice measured. The measurementof fat wa done by the follo ing recovered to almost normal condition.The LD values are general method, but to do more complete extraction theShown in table that is g-lkgr ma a d 3.2 g-lkgf r residue was extractedagain warm, extracted solution was colfemale. which indicates distinctdifference between the lected, concentrated, weighed. sexes.

liver TABLE 2.-TEST CONDUCTED ON MICE 5 times volume chloroform:methanol (2:1) added, trltura- Intro-abdominal in ection, male mice oftho dd-series, 212g. weight tion by homogenizer and extraction, washedwith 15 times [Number oi the alive] volume of the same solvent andfiltered Residue Days Filtrate added 10 times volume of 0 l 2 3 5 7 thesame solvent warmed and extracted on water bath 40 for 30 minutes,filtered Dose of thiocominer 100 mg. (in 1 ml.) 6/6 3/6 3/6 2/6 0/6 0/6Fmmte Residue 80 mg. (in 1 ml.), 6/6 6/6 3/6 1/6 1/6 l/6 60 mg. (in 1ml.) 6/6 5/6 5/6 4/6 3/6 3/6 40 mg. (in 1ml.)..... 6/6 6/6 6/6 6/6 6/66/6 Gathered, 20 mg. (in 1 ml.) 6/6 6/6 6/6 6/6 6/6 6/6 cone in vacuoPhysiological salt solution (1 ml)... 3/3 3/3 3/3 3/3 3/3 3/3 driedweighed No'rE: LD =3.0 g./kg. calculated by the Behrens-Kurber method.

a Result: Intra-abdominal injection, female mice of dd-series, 19.7 g.weight TABLE 1 [Number of the alive] Contained Liver Fat weightIncreasing weight weight of fat ratio of fat li (g.) (g.) (percent)(percent) 0 1 2 3 5 7 Dose of thiocomine:

100 mg. (in 1 ml.) 80 mg. (in 1 ml.) mg. (in 1 ml.) 40 mg. (in 1 ml.)

20 mg. (in 1 ml.) Physiological salt solution (1 ml.) 3/3 3/3 3/3 3/33/3 3/3 Average 5. 38 0. 3152 Fattly liver group:

Nora: LD =3.2 g./kg.

Acute Toxicity Test of Thiamamidine Phosphoric Cysteine Average Theresults of the acute toxicity test of thiamamidine Control group.phosphoric cysteine when applied to mouse are as follows:

1. Procedure 2 0 2729 3 0.2210 1. Animals: Male and female mice of thedd-series were 4 r s-swa 8 852 8 used. They had been fed with solid foodand tap water and 5 0'2573 2 were forced to abstain from food and waterfrom 3 hours on Average 5. 0.2413 0 before the administration ofthiamamidine phosphoric Increasing ratio or m: cysteine. After theadministration, general symptoms were ob- Average of thiocom ngr0up-8verage Ofoomwl E D served, while the dead members were dissectedto examine Average of fatty liver group-average of control group Theresults of the acute toxicity test of the inventive substance thiocomineare set forth infra.

7 within 72 hours.

2. Administration and dose: lntra-abdominal injection (i.p.) was givento male and female mice. Each 1 ml. of physiological salt solutionswhich contain 90, 70, 50, 30 and 10 mg. per individual animal wasinjected and, on the other hand, 1 ml. of physiological salt solutionalone to a control group. A group of 6 members of the animals was usedfor each dose and 3 for the control group.

Results The general symptoms such as degraded movement, erected hair,increase of the respiration frequency, etc. were noticed at 30 minutesonward after the administration by intra-abdominal injection, where themore the dose, the more remarkable the symptoms. All the members whowere led to death died at 848 hours after the administration, while theremainder of the animals gradually recovered and almost recovered theirnormal condition in 48 hours. Further, no remarkable abnormality wasnoticed in any organ of the dead members by visual observation. The LDvalues were 2.9 and 3.0 gJkg. for the male and the female, respectively,and no significant difference was observed between male and female.TABLE 3.ADMINISTRATION OF THIAMAMIDINE PHOS- PHORIC- CYS'IEINE TO MICE(a) Intra-abdorninal injection, mice of the dd-series; male; 19.5 g.weight [Ratio of the alive to the total] Dose:

90 mg. (1 m1.) 6/6 /6 0/6 O/6 0/6 0/6 70 mg. (1 ml.) 6/6 2/6 2/6 2/6 2/62/6 50 mg. (1 ml.) 6/6 4/6 4/6 4/6 4/6 4/6 30 mg. (1ml.) 6/6 5/6 5/6 5/65/6 5/6 mg. (1 mi.) 6/6 6/6 6/6 6/6 6/6 6/6 Physiological salt solution(1 ml.) 3/3 3/3 3/3 3/3 3/3 3/3 Nora: LD =2.9 g.[kg. as calculated bythe Behrens-Karber method.

(b) Inna-abdominal injection, mice of the dd-series; female; 18.8 g.weight [Ratio of the alive to the total] Days Dose:

90 mg. (1 ml.) 6/6 O/6 0/6 0/6 0/6 0/6 70 mg. (1 ml.).. 6/6 3/6 2/6 2/62/6 2/6 50 mg. (1 ml). 6/6 4/6 4/6 4/6 4/6 4/6 30 mg. (1ml.) 6/6 5/6 5/65/6 5/6 5/6 10 mg. (1 ml.) 6/6 6/6 6/6 6/6 6/6 6/6 Physiological saltsolution (1 ml.) 3/3 3/3 3/3 3/3 3/3 3/3 NOTE: LD =3.0 g./kg.

SUMMARY organ of the dead members by visual observation. So far as the icalculated values of LD are concerned, no significant difference wasobserved between male and female.

As examples of this invention the outline of the method of isolationwill be described below.

EXAMPLE 1 C. for several hours, during which period glucoside as well ascondensation part hydrolyzing enzymes, or in other words enzymes whichattach glucoside of perfume ingredients with sugar and the condensationpart of perfume ingredients with peptide, were entirely decomposed. Whenthe perfume ingredients were needed, they were collected by steamdistillation or extracted with such solvents as hexane, petroleum, etc.After the perfume ingredients had been removed, the remainder was usedas the raw material of this invention. In the following example thescale was for 10 kg. of the material. The material contained in general60-70 percent water. Either without any pretreatment or after beinggently heated to expel most water, it was placed in a vessel equippedwith a stirring device, and an adequate amount of methanol was added sothat the content of methanol was close to 60 percent. The whole mixturewas stirred for several hours, particularly while at 3540 C. in winter.Contaminate matters in the leached liquid were removed bycentrifugation, and the remaining solution was subjected to filtrationto obtain a dark brown transparent filtrate. It was then evaporateduntil specific gravity l.l-l .12 was reached and hydrolysis treatmentwas carried out with 2 liters of 28 percent sulfuric acid added for 10hours in the usual process with superheated steam. Humin substance wasformed as black precipitate which was filtered off. To the resultingsolution, first barium carbonate and secondly barium hydroxide wereadded, taking care of the bubbles of carbon dioxide, to completelyremove sulfuric acid, and the remaining dark brown solution of slightlyacidic reaction was evaporated under reduced pressure to specificgravity 1.15-1.20. To the concentrated solution 1.8-2.0 kg. of activecharcoal was added and the mixture was cooled with water under stirringfor 10 hours in which period the whole T.P.C. was adsorbed onto thecharcoal. The charcoal was filtered off to separate it from the filtratecontaining thiocornine.

The desorption of T.P.C. from charcoal proceeded in such a way that thecharcoal and 34 liters of methanol were placed in a vessel equipped witha countercurrent cooler and treated below 60 C. for 10 hours. When thesolvent was recovered, 5-6 g. of syrup containing crude T.P.C. wasobtained. Since a small amount of sugar and amino acids as impuritieswas found in the syrup, it was dissolved Uflldfil' grinding into puremethanol, and the solution was warmed with active charcoal at about 60C. for decoloration. The methanolic solution was evaporated underreduced pressure at a temperature below 60 C. under nitrogen bubbling,when crystals of T.P.C. separated gradually. The whole mixture was keptstanding in an ice room for 1-2 days to secure complete crystallization.The crystals were in plates or prisms and the yield was 5-6 g. Theclosed-ring form had the maximum ultraviolet absorption at 275 or 278 t,while the open-ring form did not absorb. The reactions they exhibitedwere 1. Prebludas reaction 2. Weyls reaction 3. Sakaguchis reaction(creatine and thiazol reactions) The open-ring form showed, in additionto the above, a reduction reaction in which methylene blue wasdecolorized in a slightly alkaline solution, and a precipitationreaction in which it formed a yellow precipitate of hydrazone byreaction with Bladishsche reagent (hydrazine reagent). The two forms ofthe compound were separated from each other by fractional dissolutionwith ethanol.

The filtrate from the active charcoal contained thiocomine. Thiocominein it might be separated from those amino acids that had been producedby the decomposition of peptide by precipitating it as phosphotungsticacid salt or other heavy metal salts because thiocornine was the onlybasic amino acid, but the process was expensive and was not free fromappreciable loss so that the following alternative method was employed.

At first the filtrate was concentrated by evaporation to specificgravity 1.2-1.25 (60-70 C. under reduced pressure) and the pH wasadjusted to 10-11 by adding caustic soda (or ammonia) under cooling andthen adding of an adequate amount of ethanol produced curdy precipitate.Further addition of ethanol in an adequate quantity under stirring andstill standing cool for a while, produced shapely needles or prisms ofcrystal. But since the free form is unstable and liable to be decomposedin a short time, the crystals were collected without delay and dissolvedin dilute hydrochloric acid. The resulting solution, when evaporatedgently, separated thiocornine hydrochloride. The yield was 1.2-1.5 g.The ninhydrin reaction exhibited characteristic reddish yellow toreddish orange.

in addition to the above examples, the same process could be applied tobeer pressed cake, bran of rice and wheat, residue of soy bean, etc.,where these matters could be used without enzymatically decomposingglucoside of perfume ingredients, because they do not smell so badly asthe Genus alliurn.

EXAMPLE 2 Process for preparing from materials of animal source (fromorgans of cattle, whales and other fish as well as skimmed milk).

In this case except for skimmed milk, organs were ground and aged at 380C. for several hours for the purpose of enzymatic decomposition,especially of lypase action, then grease was removed with a solvent bythe usual process. The degreased material was then treated with 60percent methanol. The solvent methanol was recovered from the extractsolution and the residue was brought into an aqueous solution. Theingredients were decomposed with sulfuric acid, and after the acid wasremoved the resulting solution was treated with active charcoal. By thesame process as was described in the foregoing example, T.P.C. andthiocomine were collected from the charcoal adsorbent and the filtrate,respectively. The yield, which varied depending on the animal speciesand the locality, was greater than that from plant and was -15 g. and3-5 g. for T.P.C. and thiocomine, respectively.

Alternatively the yield of T.P.C. and thiocomine in the process in whichlactic acid was cultivated and the bacteria was used was in between theyields from animal and plant sources, that is 7-8 g. for T.P.C. and 2-4g. for thiocomine.

EXAMPLE 3 Extraction from garlic, onion and the like.

Scordinine is subjected preliminarily to partial hydrolysis by 20percent sulfuric acid or percent (4N) hydrochloric acid andthiocornyl-thiamamidine phosphoric acid cystine (called thiamacornine)is then isolated from the hydrolysate.

Partial hydrolysis Scordinine I |IH 1111i ([711 HgN-(E-NH-S-CHg-CHgOH NH/C=CGH,CH OH EXAMPLE 4 Extraction from milk casein or soya bean protein.Direct isolation from the protein.

The protein is subjected to total hydrolysis by 6N hydrochloric acid or30 percent sulfuric acid. The hydrolyzate is then freed from the acidand concentrated by evaporation at low temperature under a substantiallyreduced pressure until it is concentrated into a syrupy matter having aspecific gravity of 1.25-1.30. On the addition of ammonia or causticalkali to this syrupy matter till a pH value of 4.5-4.0 is established,there is produced a large amount of precipitate, which contains besidesthiocomine small amounts of other amino acids and about 20 percent ofcommon salt (derived from the hydrochloric acid employed in thehydrolysis). l-lereupon, the precipitate is dissolved in water (underwarming) so that the concentration of the resulting solution may beestimated as about 30 percent and the solution is then left standing ina cold place, almost all the common salt being thereby crystallized.After removing the common salt crystals by filtration, the filtrate issubjected to gentle evaporation under reduced pressure, crystals ofthiocomine being thereby separated. When the thiocomine isrecrystallized from water, thiocomine dihydrochloride is obtained in acrystalline state (it is possible if desired to obtain thiocomine in theform of picrate).

EXAMPLE 5 Extraction from milk casein or soya bean protein.

Direct isolation from the protein.

The protein is subjected to total hydrolysis by 6N hydrochloric acid.The hydrolyzate is freed from the acid and other impurities inaccordance with the routine method, decolorized and concentrated underreduced pressure until the concentrate shows a specific gravity of1.30-1.28. When the concentrate thus obtained is brought to pH 4.5-6.0by the addition of caustic alkali, there is produced a large amount ofprecipitate which contains common salt derived from the hydrochloricacid employed and small amounts of other amino acids. The precipitate isdissolved in water under warming, of which the quantity is such that anaqueous solution is produced which has a concentration of about 2530percent. After the removal of insoluble matters by filtration, thefiltrate is left standing in a cold place, a large amount of common saltcrystals coming out of the solution. After removing the com mon saltcrystals by filtration, the filtrate is gently evaporated under reducedpressure, the best part of the common salt being thereby once morethrown out of the solution (the saturated solution of common salt has aconcentration of about 30 percent). The common salt crystals are removedagain by filtration and the filtrate is concentrated by evaporationunder reduced pressure, dihydrochloride of the thiocomine being therebylet out of the solution. When the thiocomine dihydrochloride isrecrystallized from water, the final product which is of l-form and hasa [01],, of -l2.5 is obtained as octahedral crystals.

Scordinine A, parent substance of T.P.C. can be grouped into a closedtype and an open type. The closed type Scordinine A and the open typeScordinine A can be separated from each other since the closed type issoluble in ethanol while the open type is soluble in a solvent ofmethanol and water 1:1). Each of thus separated closed type and opentype Scordinines is hydrolyzed and then subjected to fractionaldistillation to obtain closed type T.P.C. and open type T.P.C.

What is claimed is:

l. A new amino acid, thiocomine, having the following chemicalstructure:

and pharmaceutically acceptable acid salts thereof.

dihydrochloride.

4. A compound according to claim 1 which is thiocornine picrate.

2. A compound according to claim 1 which is thiocornine.
 3. A compoundaccording to claim 1 which is thiocornine dihydrochloride.
 4. A compoundaccording to claim 1 which is thiocornine picrate.